Proc. Natl. Acad. Sci. USAVol. 88, pp. 7242-7246, August 1991Neurobiology

Molecular characterization of a protein-tyrosine-phosphataseenriched in striatum

fb&mg la/nolcur donig

PAUL J. LOMBROSO*t, GEOFFREY MURDOCHtt, AND MICHAEL LERNERt*Child Study Center, tSection of Molecular Neurobiology and Howard Hughes Medical Institute, Yale University School of Medicine, 330 Cedar Street, NewHaven, CT 06510

Communicated by Charles F. Stevens, April 2S, 1991

ABSTRACT A cDNA clone encoding a neural-specificputative protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) has been isolatedfrom a rat striatal eDNA library. The deduced amino acidsequence predicts a protein of -369 amino acids with a ftrenghomology to other members of the family of protein-tyrosine-phosphatases. In vitro trauslatlon produces a protein with anapparent molecular mass of 46 kDa. A potential attachmentmechanism to the cytoplasm membrane is suggested by amyristoylation amino acid-consensus sequence at the N termi-nus ofthe protein. RNA analyses ofvarious regions ofrat brainreveal a 3-kilobase (kb) and a 4.4-kb mRNA. The 3-kb mRNAis highly enriched within the striatu relative to other brainareas and has been termed a "striatum enrichedphosphatase"(STEP). In contrast, the 4.4-kb message is most abundant inthe cerebral cortex and rare in the striatum. These twomessages appear to be alternatively processed RNA transcriptsof a single gene.

It is well established that modulation of tyrosine phosphor-ylation is one mechanism for controlling cellular proliferationand differentiation (1). The addition and removal of phos-phate groups on tyrosine residues is thought to be tightlycontrolled by the opposing actions of protein-tyrosine ki-nases and protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48; PTPases) (forrecent review, see ref. 2). Until recently, research hasconcentrated on the role of protein-tyrosine kinases in cel-lular control mechanisms.

Originally, phosphatases were viewed as a small group ofenzymes with a rather broad substrate specificity. The recentisolation of a number of PTPases suggests that this may notbe correct. PTPases were first identified when the humanplacental PTPase 1B was isolated, purified, and shown to behomologous to a repeated sequence in the cytoplasmic do-main of the leukocyte cell-surface protein CD45 (also knownas leukocyte common antigen, or LCA) (3). Although thefunction of CD45 was not known, the fact that CD45 andPTPase 1B were homologous suggested that in lymphocytes,CD45 might regulate the state of phosphorylation of a groupof substrate proteins. Subsequent experiments confirmedthat PTPases dephosphorylate tyrosine residues, with little orno effect on serine/threonine residues (4, 5).

Since the initial report on PTPase 1B, a number ofPTPaseshave been cloned and their amino acid sequences deter-mined. Two basic variants have emerged. Those in the firstclass have extracellular domains linked to cytoplasmic, cat-alytic domains through single transmembrane regions. ThesePTPases may interact with soluble factors, surface moleculeson neighboring cells, or extracellular matrix molecules. Sev-eral members of this first class ofPTPases have been isolated

(6-11). PTPases in the second class are located intracellularlyand lack a transmembrane or extracellular domain. Severalhave been isolated and characterized as well (5, 12, 13).Northern analyses of different tissues indicate a wide

variability in the regional expression of PTPases. For exam-ple, one transmembrane PTPase has been found in all tissuesand cell lines tested, suggesting a ubiquitous role (11).Meanwhile, others have been identified as present in a limitednumber of tissues and cell lines. Finally, another PITPase(CD45) is expressed only in cells of hemapoietic lineage (14).However, none of the PTPases identified to date have beenshown to be nervous system specific.Here, we report the isolation and characterization of a

cDNA clone that encodes for a putative PTPase foundpredominantly in the brain.1 Within the brain, this PTPaseshows further tissue specificity by appearing to be a "stria-tum enriched phosphatase" and has been designated STEP.This PTPase contains regions homologous to ones found inpreviously characterized PTPases and appears to be a distinctmember of the cytoplasmic class of PTPases.

MATERIALS AND METHODSRNA Isdation. Total RNA from bovine putamen was

isolated by using a modification (15) of the guanidiniumisothiocyanate procedure of Glisin et al. (16). For libraryconstruction, poly(A)+ RNA was twice selected by affinitychromatography using oligo(dT)-cellulose (17). For Northernanalyses, poly(A)+ RNA from rat organs and dissected brainregions was selected once using oligo(dT)-cellulose (FastTrack, Invitrogen).

Subtractive Putamen cDNA Library. First-strand cDNAwas synthesized from 5 /kg ofbovine putamen poly(A)+ RNAusing mouse Moloney leukemia virus reverse transcriptase(Boehringer Mannheim) (18) and was hybridized in the pres-ence of 40 Ag of poly(A)+ RNA isolated from bovine cere-bellum in 0.125% SDS/2 mM EDTA/0.3 M phosphate bufferin a final volume of 20 IAI (19). The sample was covered withparaffin oil, denatured at 90TC for 30 sec, and hybridized at650C for 24 hr. The sample was passed over a hydroxylapatitecolumn (HAP), and single-stranded cDNA was eluted with0.12 M phosphate buffer/0.1% SDS, and a second cycle ofsubtractive hybridization was performed. Second-strandcDNA was synthesized by usingDNA polymerase I (Klenowfragment) (20, 21), followed by S1 nuclease digestion (21) andC-tailing (22). The plasmid pUC-9 (Stratagene) was linearizedat its Pst I site and G-tailed to allow hybridizing to C-tailedcDNA.

Abbreviations: PTPase, protein-tyrosine-phosphatase; STEP, stria-tum enriched phosphatase.*Present address: Division of Neuropathology, Oregon Health Sci-ences University, Portland, OR 97201.§The sequence reported in this paper has been deposited in theGenBank data base (accession no. M65159).

7242

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Aug

ust 1

4, 2

021

Neurobiology: Lombroso et al.

Isolation of Additional STEP cDNAs. A randomly primedstriatal Agtll cDNA library (Clontech) was screened by usingrandomly primed [32P]dCTP (Amersham) radiolabeledprobes (23). The initial probe was a 300-base pair (bp) bovinecDNA clone. Filters were hybridized in buffer containing50% (vol/vol) formamide/Sx Denhardt's solution/5x stan-dard saline phosphate/EDTA (SSPE; lx SSPE is 0.18 MNaCl/10 mM phosphate, pH 7.4/1 mM EDTA)/1% SDS/boiled salmon sperm (100 pg/ml) at 420C for 18 hr, followedby washes in 0.5x SSC/0.1% SDS at 650C. EcoRI inserts ofpurified clones were subcloned into the plasmid vector Blue-script (Stratagene), and DNA was purified by CsCl ultracen-trifugation (24).DNA Sequence Analysis. Nucleotide sequences were ob-

tained using the chain-termination methods of Sanger (25)after either subcloning inserts into M13 bacteriophage or byusing cDNA clones in Bluescript, which were progressivelydigested using Exo III nuclease (Erase-a-Base, Promega).Sequences deposited in GenBank were searched for homol-ogy with a sequence analysis software package (University ofWisconsin).Northern Blot Analyses. Two micrograms ofpoly(A)+ RNA

was electrophoresed on 1% formaldehyde/agarose gels,transferred to nylon membrane and hybridized with ran-domly primed probe (1 x 106 cpm/ml) under high-stringencyconditions in 50% (vol/vol) formamide/5x Denhardt's solu-tion/5x SSPE/1% SDS/boiled salmon sperm (100 ,ug/ml) at42°C, and washed at 650C in 0.1x SSC/0.1% SDS. Probeswere either a 2.4-kb cDNA clone (9.6), which consists of-600 bp on either side of the open reading frame or a 400-bpApa I-Pvu II fragment within the 3' untranslated region.

In Vitro Translation of STEP. A plasmid containing thecomplete STEP open reading frame was constructed fromoverlapping partial clones by splicing at the unique BamHIsite (Fig. 5, for restriction map). This sequence was tran-scribed using T3 RNA polymerase (mRNA capping reaction,Stratagene). As a control experiment, antisense message wastranscribed using T7 RNA polymerase. Two and one-halfmicrograms was added to a rabbit reticulocyte in vitrotranslation mix (Promega) in the presence of [35S]methionine(Amersham). Twenty microliters of this reaction was loadedonto a 12% SDS/polyacrylamide gel and processed by stan-dard procedures (26).Southern Blot Analysis. Rat genomic DNA was digested

with restriction enzymes that either had no restriction sites inthe cDNA clone (Xba I and HindIII) or had a single site(EcoRI and BamHI). Five micrograms of digested genomicDNA was fractionated on an 0.7% agarose gel and, after briefacid depurination, transferred in 0.4 M NaOH onto Zeta-Probe membrane (Bio-Rad).

RESULTSIsolation and Characterization of STEP cDNA Clones. A

putamen-enriched plasmid cDNA library was constructedfrom bovine tissue. Over 90%6 contained inserts with anaverage size of300 nucleotides, and 10%6 ofthese representedmRNAs that were enriched in the putamen relative to cere-bellum (data not shown). One of these clones containing a300-bp insert was used to screen a rat striatal Agtll library.The initial screen yielded a 1-kb cDNA clone (9.2) containingportions of an open reading frame (see Fig. 5 for all clonesisolated). The 5' region was used to rescreen Agtll librariesto obtain overlapping clones encoding a complete openreading frame. The sequence obtained from these overlapping cDNA clones is shown in Fig. 1.The predicted amino acid sequence ofthe single, 1107-base

open reading frame identified the gene product as a potentialmember of the phosphotyrosine phosphatase gene family.The highly conserved 12-amino acid domain originally iden-

Proc. Nati. Acad. Sci. USA 88 (1991) 7243

SCGG rCCAT7 ccS ATIA A3GC77T GC CTAC7TAA7TCTA62 r22AACGAA222AA2 AC7SGAGrrCrcAAC 7ACA:lrCTCAGSlAA7 7AA77 r'0AT C--'-- ACCCrAACC- .ATAAATAAAAASAc- AG= A7C7SC ..T AA.,A... G- 0-AAa0OCCT7. ..CAQAC7TC:Cc

212 7TC7.ATGTGTACC. GG.A....A .... .A. .........A .A. T...CTCAA3AC

A-27GAG=AC7C7Cr CAGCAA7ATAGSGCAA..... CAA. C ..7.A, AGAAC7Ccrr

512TCTCCTCCCTC77AACCACCT222OccACCAC7GTA GTTTS-ACC2GG G2A0CAAACTC7cc

662 CCC. 7CA7 CCCCACASACASA CSCAc A7CclACc c l~OGA..... . A... ACrcAGAG=

2V'eAspCsVa peAs .,:leLy Pro Glo. Ala OAsp'rPo 7"7 Sar :.rPTr Va. 15ys er-

4e-GI :.P '; rzorg G:ySer As al Slare-lchr Asp Metcysr ,r Pru8&Z:;T CC C2 CA11A 022AGO 7CC ',AT.7 .C =. ACC C7cc cAc A: crG AC--cc

6GI~ys snO' ~ 3-2;G yPre dY :yr laVal Ser P rc Ara 2.. Sar Ala L` I9::_7 cSc Cc AA--' A2 ZAG SC 7-7-- -- S-..7.. .- .~..A GAO GAG .A ..cC CAr J2A

61 ryr LT e.- Leu Sea- A-a Se- Arg Pal Lea.. Ara Ala 2lo S21 1ao s GbI Ly Ala Leu Asp

:1: P7- Phe Leao Leao 2> Ala GI P6,ePhe 0o Il,:- Pro Met Asn Proe Va: Asp Pro Ly. GI.71:ccc Tcc ccc' Ccc Coc 11cc coo 7cr Tcc SAA Ar-c CC-C ArcA 7cccc G GA2:ccA AAA 2AA

:2: cyr Asp :le Pro Gdy Lao P.Va Arg Lys Aar Arg r'yr Lys chr Ile Le-. Pro Aso Pro Hi-s:197 cAr AcCArc rcA GGG Ccc GcA ccc oAoc oAr7CG ACoC AAA Arc Arc Ccc ccc AT -Cc-c oCA

'4: Oar Arg Val Arge-;lao cr Sar -Pro Aspo Pro SI Asp Pro Leao;Sr Sar Tyr Ile Aar Ala::515 ACC AG2 crA CST CTG Acc rCA rcA cAc ccT SSAA CAT ccc_ CTG ACT TCC 7Ac- Arc oAr CC-

:6: As- cyr :Ie Arg Sly ryr Oar. C:y do Ly.s Vac cyr :le Ala cor Gin Sly Pro e

:2:7 OAr cAr Arc COG GCc rAc AAr 2211 2A2 202 AAn Gcrc TCo Arc CCc Acc CAG CGA CCC Arc

:6: Va: Sar chr Va: Val Aor P6,e cro Org Mer~ Val rrp cc-.G2Xo Ara crr Pro :l :e a

:277 SCc Acc ArCT cc =Cc cAr TCc 'Cc cCc Arc crc ccc coc cAc cCc ACA ccc ArC Arc SCc

211 Met :le c`r Asn :le do : Met Asr Gc:. Lys Cys c6r cI r.yr crv Pro G-;S ,G:337 Arc Arc AcrC ArC Arc cAG dAd Arc AAc SAG AAG GCc ACC CAA cAr ccc rCA r.AA GAd rcA

22 Val Val HsAsu dlp Val GIu:le cor Va: 2>- 7ysaP. I s7Icr 2I>. Asp Tyr Ar:397 cc Gcc CAc cAr GCc crA GAG Arc ArCT OCc cA.c AAA C-C AoCc coc AcA GAG GCc ACc CCc

241 Le;; Org Le-. :le Oar Le, Org Org d--p cnrr o Arg dy ,~. Ly-s -'5 co cr 'Pi

261 7crr er crvc:Pr o SinLysca crcPro Asp Ora Ala P~ro P:ro lao Lao; 7-los .eao Val A

261 do_P.al d 1. Al-a Ala GdI doO do01-;SyPro shysSepOr Pro _11 Ie:e Pa I9.Hi a15577 Coc ccG GAG 202 GCc ccc cAc 11AA 202G 22cA c cAc 7Cc 7CC cr2 Arc Arc7c7~ 2

2111c~~r~~l.2~ l~~e 62~y Ar; rcA- Ply Cpa 3~~~:re Aca.'crrSer Il pacps2y r

1637 7-1cc~~PC02 c o -C' Arc CCc ArcC 02O Arc Tcc cccC cASCA

32: Org Org dou Gy Va: Pal Asp K le Le-. cy7r crr Cys Gin Le-. Org Sdo Asp Ora ccp1697 rCA CG ASGS OCGA CAC Arc cr0 AA C C C A 7 G A A C C

341 clp Met K, 2cr cIn r Cpa 2>. 2>n Tyr GIc Pr6e Val i HIs I Ala Met Ser Lao Tyr Si1o1757 SCc Arc Arc cA.o AcA 7cc coAA CA02:0 CAC TTT cccGCAC cAr.-G .. A... cAr coc

361 lps Gir leu Ser leo Sir Oar Scr do- "

1:7: 002 CAc cr0 TCC C7C CA2 :CC Cro 20A1 rcA Cr0 CAC cccT=Cccc ASA.5C ..A.-.,.ACTCllCS

1963 AGCTGAcc7GGGccc C7CrccCCCACooCco ccoAo0CTACCrocccc T;OC7G CCTGTCcC1956 77A 7CCC G7C-ACCAGCA77ZA GAAA7CS

2333 GC7ACCAATCTCTcccCCcCc.A.AGCATG7 GGA. .2T..AGACC.A GG,.A.AAAA.GT0.G0......... AGA

2463 cAGrGGGAAACCCcA AccA7CTco'-C-''- ACZCCTCA GAG7GG-C CCAACSA2556 ZACTCGGcccTAccccrccccccCAAA7ScccCo7CcCcTo.Aoccccccc7oocCccCCGT S7ScCCccccrTGCo2633 =77STSc7cccoAG CCCocTo...-A.....A ..-,A..o .0..... CAACA.-00....7A0.C. .,..A .C..A.-.-...

2763 ccCOAcAcAA coAF A AGAGCATT-TGA 2110(62)

FIG. 1. Complete nucleotide and deduced amino acid sequence ofSTEP. Three potential initiating codons are underlined. Polyadenyl-ylation signal is indicated by an open box. The 12-amino acidconsensus sequence [His-Cys-Ser-Ala-Gly-(Ile/Val)-Gly-Argz-(Ser/Thr)-Gly-Xaa-(Phe/Tyr)] found in all tyrosine phosphatases isolatedto date is indicated by the filled-in box. *, Stop codon.

tified by Charbonneau et al. [His-Cys-Ser-Ala-Gly-(Ile/Val)-Gly-Argy-(Ser/T'hr)-Gly-Xaa-(Phe/Tyr)] is highilighted in Fig.1 (12). A comparison of the predicted amino acid sequence ofSTEP with the previously described intracellular PTPases,T-cell PTPase and placental PTPase iB, reveals --30%o iden-tity. The degree of similarity 'increased to >50% by allowingconservative substitutions. As illustrated in Fig. 2, most ofthis similarity falls within a 250-amino acid region character-istic of the catalytic domain of all PTPases. Similar to otherintracellular PTPases, this domain is present only once. Incontrast, the transmembrane receptor-like PTPases have twophosphatase domains. Although the catalytic domain is lo-

Dow

nloa

ded

by g

uest

on

Aug

ust 1

4, 2

021

7244 Neurobiology: Lombroso et al.

PTPase 1 B E

STEP ES

T Cell PTPase

PTPase 1 B

T Cell PTPase EL

:I:!

I;,~ ~ ~

111 i

z1

]

50 AA

FIG. 2. Sequence similarities ofintracellular PTPases. The aminoacid sequences of STEP, PTPase 1B, and T-cell PTPase werecompared by pairwise Pustell matrix analysis (IBI/MacVector) usingan 8-amino acid window. Regions with a similarity score of>90% areshaded black; regions with a similarity score of >50% are stippled.

cated near the C terminus of STEP, in the other cytosolicPTPases described to date it is closer to the N terminus.The overlapping striatal cDNA clones account for 2816

bases of coding and noncoding sequence including thepoly(A) addition signal (see Fig. 1). A putative polyadenyl-ylation signal, AATAAA, was found at nucleotide 2794 andwas followed after 17 additional nucleotides by a poly(A)+ tailof 60 adenosine residues.The predicted size of the STEP protein is between 38 and

42 kDa. There are three methionines at the 5' end of the openreading frame (underlined in Fig. 1). The first of thesepotential initiator codons predicts a 369-amino acid proteinwith an approximate molecular mass of 42 kDa. The nucle-otide sequence surrounding the third methionine, however,gives a better match with the Kozak sequence for eukaryoticinitiation sites (RCCAUGG) (27). This third potential initiatormethionine predicts a 329-amino acid protein of 38 kDa. Togain insight into which of these methionines could act as theinitiation codon, in vitro RNA transcripts were translated byusing a rabbit reticulocyte translation system. A nearlyfull-length fusion cDNA under the control ofa T3 polymerasepromoter was constructed (clone 9.6). Fig. 3 shows that theresultant RNA transcripts direct the synthesis of a single

prominent protein with an apparent molecular mass of 46kDa. A few minor smaller proteins were seen with prolongedexposure. Although these results are most consistent with theuse of the first potential initiator methionine, PAGE may notbe sensitive enough to distinguish between the three sitesaccurately. Generation of antisera will be required to deter-mine the actual size ofSTEP protein in vivo (see Discussion).

Expression of STEP mRNA. The initial nucleic acid-subtraction strategy used to isolate STEP was designed toidentify gene products enriched in striatum. Fig. 4a demon-strates that STEP is selectively expressed in the nervoussystem. Whole-brain RNA contains a predominant 3-kbmRNA species and a second 4.4-kb species that is moredistinct on longer gels when probed with the 400-bp 3'fragment (Fig. 4A, Right and see Discussion.) With prolongedexposures (2 weeks), these mRNAs were barely detectable in

Ar_C Al

_0

7.5-

4.4 -

2.4-

1 .4-

4.4-

2.4-

B

1 2 3- 200k

- 93

- 69

- 46

C) " U

Z a) w1 c

, Qr > -. i

I U) L Y m

0 24-

' .4 -

2 nouar

2 veeK

I4x

x CD)

E 0 C.._ E X X

X X - O 'C m4-

m C) Co

.(a

*- co C)4

7.5 -

4.4 -

2.4 -- 30

UAid

1.4 -

28S RNA- 14 ow ::..F....

FIG. 3. In vitro translation of STEP RNA transcripts. In vitroRNA transcripts were translated with a rabbit reticulocyte lysatesystem in the presence of [35S]methionine and analyzed by 12%SDS/PAGE, as described. Lanes: 1, no added RNA; 2, 2.5 pg ofclone 9.6 sense RNA; 3, 2.5 ug of clone 9.6 antisense RNA.Autoradiogram was exposed overnight.

FIG. 4. Analysis of STEP mRNA abundance. (A) Northern blotofpoly(A)RNA isolated from various rat organs probed with a 2.4-kbfragment spanning the entire open reading frame (clone 9.6). Auto-radiograms were exposed as indicated. The blot at right was probedwith a 400-bp 3' fragment and exposed for 24 hr. (B) Northern blotof poly(A) RNA isolated from various rat brain regions and probedwith 400 bp of 3' fragment. Autoradiogram was exposed for 24 hr.

III E-- 1.1m

Proc. Nad. Acad Sci. USA 88 (1991)

1 1

Dow

nloa

ded

by g

uest

on

Aug

ust 1

4, 2

021

Proc. Natl. Acad. Sci. USA 88 (1991) 7245

adrenal, kidney, and heart. The ratio of band intensitiesdemonstrates that STEP mRNA is much more abundant inbrain relative to peripheral tissues.The distribution of STEP mRNA within the central ner-

vous system is shown in Fig. 4b. The 3-kb species is highlyexpressed in the striatum. In contrast, the 4.4-kb speciesappears to be expressed selectively outside the striatum. The3-kb mRNA is also present in other brain regions but at lowerlevels of expression. As expected from the initial subtractionstrategy, neither RNA message is present in the cerebellum.A Southern blot of rat genomic DNA is shown in Fig. 5.

Multiple genomic fragments are present with two restrictionenzymes (HindIII and Xba I), which do not cut the STEPcDNA, and with BamHI, which has a single internal site (seeFig. 5 for a restriction map). The STEP gene thus consists ofseveral exons distributed over at least 15 kb of genomicDNA.

DISCUSSIONThe identification of STEP as a PTPase is based on its highhomology to the core amino acid sequence found in otherPTPases isolated to date, but specific phosphatase activityhas not yet been demonstrated. Although a number of otherPTPases are expressed both in peripheral tissues and in brain,STEP is the only one to be highly expressed in the nervoussystem. Northern analyses reveal that the intracellular phos-phatases PTPase 1 and T-cell PTPase are expressed in brain.PTPase 1 mRNA shows a higher level of expression inlymphoid tissue (spleen) relative to brain and is present inliver and kidney (5). T-cell PTPase is more abundant inspleen, thymus, and placenta relative to brain (13). SeveralPTPases with transmembrane and extracellular domainshave also been isolated from brain cDNA libraries, althoughthese proteins are expressed by a variety of tissues (9, 10). Inaddition, biochemical fractionation reveals seven distincttypes of PTPase activity, although none have been shown tobe brain specific (28).Our results demonstrate that STEP expression is restricted

primarily to the nervous system. The small amounts oftranscripts present in peripheral tissues may actually belocalized to postganglionic autonomic neurons present inperipheral tissues, and immunocytochemical staining will beused in future studies to clarify this issue. Northern analysisof brain poly(A)+ RNA reveals two distinct signals at =3 kband 4.4 kb. The smaller band represents the mRNA thatencodes STEP. This mRNA is present in the striatum and, inlesser amounts, in the cortex and midbrain regions. In

contrast, the 4.4-kb message is present in all brain areas inwhich the lower band is found except for the striatum, whereit is not visible. Neither band is present in the cerebellum; thisobservation is not surprising because cerebellar mRNA wasused as driver in the original subtractive hybridization pro-cedures. The absence of STEP within the cerebellum alsosuggests that the cerebellum may express different or uniquephosphatase(s). This hypothesis is further supported by thefindings that the rat brain contains high levels of tyrosine-specific protein kinase activity in a regional distribution.Interestingly, the cerebellum demonstrated one ofthe highestlevel of protein-tyrosine kinase activity (29), further suggest-ing the possibility of cerebellum-specific PTPases.

Several lines ofevidence strongly suggest that the 3-kb and4.4-kb poly(A)+ species are alternatively processed RNAtranscripts derived from a single gene. (i) The same patternof bands was obtained on a Northern (RNA) blot whenprobes were used that were a nearly full-length cDNA cloneor one encompassing a region from the 3' noncoding region.(ii) This observation is also supported by the results shown inFig. 4, in which the relative abundance of the two species ofRNA on Northern blots is approximately equal when probedwith cDNA sequence complementary to the 3' region. Incontrast, the 3-kb RNA species appeared more abundantwhen the blots were probed with the nearly full-length STEPcDNA (Fig. 4a). (iii) In addition, Southern blots revealed twobands when rat genomic DNA was digested with EcoRI andprobed with the nearly full-length cDNA clone. This patternis consistent with the single EcoRI restriction site that existswithin the open reading frame of the 3-kb message. Moredetailed direct characterization of the gene structure is nec-essary to define the precise relationship between the 3-kb and4.4-kb species and to rule out the alternative possibility thatadjacent, duplicated genes encode the two mRNAs.The striatal STEP transcript generated an in vitro transla-

tion product with a 46-kDa apparent molecular mass. A369-amino acid protein is predicted if the first methioninewere used as the initiator codon. Kozak consensus rulespredict, however, that the third methionine of the openreading frame should be a better initiator codon, and thiswould produce a protein of 329 amino acids. The resultingpolypeptide would have a potential N-terminal myristoyla-tion site similar to those described for other proteins (30). Inthis situation, the initiating methionine residue is removed,and the a amino group of the N-terminal glycine is modifiedby the covalent attachment of a myristoyl group. A similarmodification has been described for a number of otherproteins, including the tyrosine kinases pp60vsrc (31) and the

_ _0.0 E c~ m -aax m x uw

23 -

Pv H E B HI I I I I

H PvI I

Bovine clone

9.2 clone

9.3 clone

9.4 clone

9.5 clonem 9.6 clone

FIG. 5. Restriction enzyme analysis of STEP cDNAs and genomic Southern analysis. (Left) Genomic Southern blot of rat genomic DNAdigested with the indicated enzymes and probed with the 9.6 clone. E, EcoRI; B, BamHI; H, HinfI; Pv, Pvu II. (Right) Restriction map of striatalSTEP mRNA (box indicates location of open reading frame) and distribution of the individual isolated cDNA clones.

9.4 -

6.5 -

Pv

2.32.0 -

ISTEP 2816 bases0 1 1 1 I I I

N

Neurobiology: Lombroso et al.

L

Dow

nloa

ded

by g

uest

on

Aug

ust 1

4, 2

021

7246 Neurobiology: Lombroso et al.

serine/threonine protein phosphatase, calcineurin (32). Al-though myristoylation could be the mechanism for membraneattachment ofSTEP, this hypothesis will need to be tested bythe addition of radiolabeled myristoyl groups in the appro-priate translation system. Polyisoprenylation or palmitoyla-tion at the C terminus of the cytoplasmic PTPases has beenreported as an alternative mechanism (5). Both mechanismswould allow for the association of these enzymes with theirsubstrates through the preexisting assembly of componentparts in the form of complexes. A similar complex has beensuggested for the epidermal growth factor receptor, a tyro-sine kinase that forms a complex with one of its substrates,phospholipase C-y (33).

In conclusion, STEP is a protein containing sequence witha high degree of homology to the core phosphatase regionfound within other PTPases and which shows enrichmentwithin the brain and a high degree of specificity within thestriatum. A number of phosphoproteins are known to beenriched within the striatum (34), including the dopamine andcAMP-regulated phosphoprotein DARPP-32 (35). DARPP-32itself is probably not a substrate for STEP, as it is phospho-rylated on serine and threonine residues (34). The identifi-cation of STEP will now potentially allow us to determinewhether there are other striatal-specific phosphoproteins thatmight serve as substrate(s) for STEP. It seems likely thatfuture studies will reveal other PTPases with patterns ofnervous system expression distinct from STEP. This degreeof PTPase heterogeneity in the central nervous system couldprovide an important mechanism for the regional control ofevents, such as cell division and differentiation.

We thank Saumyendar Sarkar for his assistance with some exper-iments, Janice Naegele and Randy Blakely for assistance with ratbrain dissections, Susan Staurovsky for technical assistance, andSusan Amara and Janice Naegele for comments on the manuscript.This research was supported, in part, by an Alcohol, Drug Abuse,and Mental Health Administration Scientist Development Award forClinicians MH00856-02 (P.J.L.) and the Howard Hughes MedicalInstitute (M.L.).

1. Hunter, T. & Cooper, J. A. (1985) Annu. Rev. Biochem. 54,897-930.

2. Alexander, D. (1990) New Biologist 2, 1049-1062.3. Charbonneau, H., Tonks, N. K., Walsh, K. A. & Fischer,

E. H. (1988) Proc. Natl. Acad. Sci. USA 85, 7182-7186.4. Tonks, N. K., Charbonneau, H., Diltz, C. D., Fischer, E. H.

& Walsh, K. A. (1988) Biochemistry 27, 8695-8701.5. Guan, K., Haun, R. S., Watson, S. J., Geahlen, R. L. &

Dixon, J. E. (1990) Proc. Natl. Acad. Sci. USA 87, 1501-1505.6. Streuli, M., Krueger, N. X., Hall, L. R., Schlossman, S. F. &

Saito, H. (1988) J. Exp. Med. 168, 1553-1562.

7. Streuli, M., Krueger, N. X., Tsai, A. Y. M. & Saito, H. (1989)Proc. Nat!. Acad. Sci. USA 86, 8698-8702.

8. Matthews, R. J., Cahir, E. D. & Thomas, M. L. (1990) Proc.Natl. Acad. Sci. USA 87, 4444 4448.

9. Sap, J., D'Eustachio, P., Givol, D. & Schlessinger, J. (1990)Proc. Nat!. Acad. Sci. USA 87, 6112-6116.

10. Kaplan, R., Morse, B., Huebner, K., Croce, C., Howk, R.,Ravera, M., Ricca, G., Jaye, M. & Schlessinger, J. (1990) Proc.Natl. Acad. Sci. USA 7, 7000-7004.

11. Jirik, F. R., Janzen, N. M., Melhado, I. G. & Harder, K. W.(1990) FEBS Lett. 273, 239-242.

12. Charbonneau, H., Tonks, N. K., Kumar, S., Diltz, C., Har-rylock, M., Cool, D., Krebs, E. G., Fischer, E. H. & Walsh,K. A. (1989) Proc. Natl. Acad. Sci. USA 86, 5252-5256.

13. Cool, D. E., Tonks, N. K., Charbonneau, H., Walsh, K. A.,Fischer, E. H. & Krebs, E. G. (1989) Proc. Natl. Acad. Sci.USA 86, 5257-5261.

14. Barclay, A. N., Jackson, D. I., Willis, A. C. & Williams, A. F.(1987) EMBO J. 6, 1259-1264.

15. MacDonald, R. J., Swift, G. H., Przybyla, A. E. & Chirgwin,J. M. (1987) Methods Enzymol. 152, 219-227.

16. Glisin, V., Crkvenjakov, R. & Byus, C. (1974) Biochemistry 13,2633-2637.

17. Aviv, H. & Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69,1408-1412.

18. Krug, M. S. & Berger, S. L. (1987) Methods Enzymol. 152,316-325.

19. Alt, F., Kellem, R., Bertino, J. & Schimke, R. (1978) J. Biol.Chem. 252, 1357-1370.

20. Maniatis, T., Fritisch, E. F. & Sambrook, J. (1982) MolecularCloning:A Laboratory Manual (Cold Spring Harbor Lab., ColdSpring Harbor, NY).

21. Gubler, U. (1987) Methods Enzymol. 152, 325-329.22. Eschenfeldt, W. H., Puskas, R. S. & Berger, S. L. (1987)

Methods Enzymol. 152, 337-342.23. Feinberg, A. & Vogelstein, B. (1984) Anal. Biochem. 137,

266-267.24. Miller, H. (1987) Methods Enzymol. 152, 145-170.25. Sanger, F. & Coulson, A. (1975) J. Mol. Biol. 9, 444-448.26. Laemmli, U. K. (1970) Nature (London) 227, 680-685.27. Kozak, M. (1986) Cell 44, 283-292.28. Jones, S. W., Erikson, R. L., Ingebritsen, V. M. & Ingebrit-

sen, T. S. (1989) J. Biol. Chem. 264, 7747-7753.29. Hirano, A., Greengard, P. & Huganir, R. L. (1988) J. Neuro-

chem. 50, 1447-1455.30. Towler, D. A., Gordon, J. I., Adams, S. P. & Glaser, L. (1988)

Annu. Rev. Biochem. 57, 69-99.31. Garber, E. A., Krueger, J. G., Hanafusa, H. & Goldberg,

A. R. (1983) Nature (London) 302, 161-163.32. Aitken, A., Cohen, P., Santikarn, S., Wiliams, D. H., Calder,

A. G., Smith, A. & Klee, C. B. (1982)FEBS Lett. 150, 314-318.33. Margolis, B., Bellot, F., Honegger, A. M., Ullrich, A., Schles-

singer, J. & Zilberstein, A. (1990) Mol. Cell. Biol. 10, 435-441.34. Hemmings, H. C., Nairn, A. C., McGuinness, T. L., Huganir,

R. L. & Greengard, P. (1989) FASEB J. 3, 1583-1592.35. Hemmings, H. C., Jr., Nairn, A. C., Aswad, D. W. & Green-

gard, P. (1984) J. Neurosci. 4, 99-110.

Proc. Nad. Acad Sci. USA 88 (1991)

Dow

nloa

ded

by g

uest

on

Aug

ust 1

4, 2

021